PROJECT SUMMARY Mammalian terminal erythropoiesis has been well studied in erythroid lineage commitment and various factors involved in differentiation and proliferation. Relatively little is known in the final steps of terminal differentiation, specifically from enucleation to reticulocyte maturation into red blood cells. Several outstanding questions in this field include what factors regulate the exit of last mitosis of erythroblast for enucleation, how nascent reticulocyte separate from the extruded nucleus, and what signals are involved in regulating the clearance of organelles in reticulocytes. Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies of in vitro or ex vivo generation of red blood cells in transfusion medicine. In this respect, we have been working on the role of mDia formins in terminal erythropoiesis and our recent novel unpublished findings may help shed light on the clues to these questions. Using a mDia2 hematopoietic-specific knockout mouse model, we revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, primarily due to the disrupted actin profilaments. mDia2 deficient reticulocytes also show defects in membrane remodeling and organelle clearance. We further revealed in our preliminary data that Chmp5, one of the major components of the ESCRT III complex, is a novel downstream mediator of mDia2. Our preliminary mechanistic studies indicated that mDia2 regulates the transcriptional activity of serum response factor (SRF) and Chmp5 is a potential novel target of SRF. These results led us to hypothesize that mDia2 regulates the ESCRT III complex to control membrane remodeling and organelle clearance during reticulocyte formation. In this project, we will use in vivo mouse models, transplantation assays, various biochemical assays, and cutting-edge microscopy to study 1) the functional roles of mDia2 in the formation and organelle clearance of reticulocytes. 2) the mechanism of mDia2-SRF- Chmp5 signaling in the regulation of reticulocyte membrane remodeling and organelle clearance, and 3) the post-translational modifications of mDia2 in its roles in reticulocyte maturation. Successful completion of our proposed studies has the potential to open a new field in signaling transduction that modulates enucleation to reticulocyte formation. Understanding the emerging roles of mDia2 as a master regulator of the late stage terminal erythropoiesis will have an important impact in this field as a paradigm.